The simplicity of Anza restriction enzymes

Anza restriction enzymes are used in a simple two-step protocol, regardless of the number of restriction enzymes in your reaction or the type of DNA you’re using—just prepare your reaction mixture and incubate at 37°C for 15 minutes.

Simple protocol

Prepare a reaction mix by adding reagents in the order indicated in Table 1.

Incubate at 37°C for 15 minutes.

Table 1:

Reagent

1-enzyme

2-enzyme

3-enzyme

Nuclease-Free Water

As required to make up final reaction volume

Anza™ Buffer (10X) or Anza™ Red Buffer (10X)

2 µL

2 µL

3 µL

DNA

0.2–1 µg

0.2–1 µg

0.2–1 µg

Anza restriction enzyme 1

1 µL

1 µL

1 µL

Anza restriction enzyme 2

—

1 µL

1 µL

Anza restriction enzyme 3

—

—

1 µL

Final reaction volume

20 µL

20 µL

30 µL

Volumes can be scaled up linearly to 5X.

Simple numbering system

Anza restriction enzymes are named with a combination of the familiar enzyme name and a number.

Restriction enzymes that are more frequently used have a lower number, so you can easily sort, store, and find the enzymes you need, without having to remember more difficult enzyme names when storing alphabetically.

Sort and store by number

Convenient buffer formats

All Anza restriction enzymes come with an Anza 10X clear buffer and an Anza 10X red buffer to give you the flexibility you require. The red buffer includes a density reagent containing red and yellow tracking dyes that migrate with 800 bp DNA fragments and faster than the 10 bp DNA fragments, respectively, in a 1% agarose gel. This eliminates tedious dye addition steps prior to gel loading and is compatible with downstream applications.*

*For applications that require analysis by fluorescence excitation, Anza 10X buffer is recommended, as the Anza 10X red buffer may interfere with some fluorescence measurements.

Digestion of plasmid DNA

Figure 2. Anza restriction enzymes are formulated to complete digestion in just 15 minutes. Plasmid DNA (6,215 bp) was digested using Anza restriction enzymes 3 BcuI and 47 Eco521, as well as Competitor N SpeI-HF (isoschizomer to BcuI) and Competitor N EagI-HF (isoschizomer to Eco52I). Reaction mixtures included 1 µg of DNA and 1 µL of Anza restriction enzyme to a total volume of 20 µL, following the recommended protocol. Reaction mixtures included 1 µg of DNA and 1 µL of Competitor N restriction enzyme to a total volume of 50 µL, following the manufacturer’s protocol. Incubation was performed at 37°C for 15 minutes for both Anza restriction enzymes and competitor N EagI-HF, as per the protocol. Incubation was done at 37°C for 5 minutes for Competitor N SpeI-HF, per the manufacturer’s protocol.

L – 1 Kb Plus DNA Ladder

C – undigested plasmid DNA

1 – Anza 3 BcuI

2 – SpeI-HF (Competitor N)

3 – Anza 47 Eco52I

4 – EagI-HF (Competitor N)

Digestion of PCR DNA

Figure 3. Anza restriction enzymes are formulated to complete digestion in just 15 minutes. Purified PCR product (1.6 kb) was digested using Anza restriction enzymes 3 BcuI, 9 NdeI, and 47 Eco521, as well as Competitor N SpeI-HF (isoschizomer to BcuI), Competitor N NdeI, and Competitor N EagI-HF (isoschizomer to Eco52I). Reaction mixtures included 1 µg of DNA and 1 µL of Anza restriction enzyme to a total volume of 20 µL, following the recommended protocol. Reaction mixtures included 1 µg of DNA and 1 µL of Competitor N restriction enzyme to a total volume of 50 µL, following the manufacturer’s protocol. Incubation was done at 37°C for 15 minutes.

L – 1 Kb Plus DNA Ladder

C – undigested DNA

1 – Anza 3 BcuI

2 – SpeI-HF (Competitor N)

3 – Anza 9 NdeI

4 – NdeI (Competitor N)

5 – Anza 47 Eco52I

6 – EagI-HF (Competitor N)

Some restriction enzymes can exhibit star activity, or a decrease in specificity to their DNA recognition site, with prolonged digestions. Star activity results in non-specific cleavage of DNA and can occur when reaction conditions are not optimal, such as high glycerol content or presence of Mg2+. Anza restriction enzymes, in conjunction with the Anza buffer, have been optimized to allow for flexibility in digestion times—from complete digestion in 15 minutes to overnight digestions—without the worry of star activity.

Anza restriction enzymes allow for complete digestion with multiple enzymes in a single reaction, using the single Anza buffer. Forget the frustrations of trying to find compatible buffers for all your enzymes, and save time with just one protocol for all digestions.

Double digestion

Figure 5. Anza restriction enzymes show complete digestion with two enzymes in a single buffer. Plasmid DNA (6,215 bp) was digested using Anza restriction enzymes 47 Eco 52I and 14 SalI. Similarly, the same plasmid DNA was digested with Competitor N EagI-HF (isoschizomer to Eco521) and Competitor N SalI-HF. Reaction mixtures included 1 µg of DNA and 1 µL of each Anza restriction enzyme to a total volume of 20 µL, following the recommended protocol. Competitor reaction mixtures included 1 µg of DNA and 1 µL of each Competitor N restriction enzyme to a total volume of 50 µL, per the manufacturer’s protocol. Incubation was done at 37°C for 15 minutes.

Figure 6. Anza restriction enzymes show complete digestion with three enzymes in a single buffer. Plasmid DNA (6,215 bp) was digested using Anza restriction enzymes 1 NotI, 16 HindIII, and 15 XmaJI. For single restriction enzyme digestions, reaction mixture included 1 µg of DNA and 1 µl of restriction enzyme to a total volume of 20 µL. Reaction mixtures included 1 µg of DNA and 1 µL of each restriction enzyme to a total volume of 30 µL for triple digestion, per the recommended protocol. Incubation was done at 37°C for 15 minutes.

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